Microbiological production of novel biosurfactants

ABSTRACT

Newly isolated microbes of the genera Arthrobacter-Corynebacterium-Nocardia as represented by Corynebacteria Salvinicum strain SFC, produce substantial quantities of materials having outstanding surfactant properties, when grown on carbon supplying substrates under fermentation conditions.

This invention relates to microbiological production of surfactantmaterials, to novel surface active materials (surfactants) ofmicrobiological origin, and to microorganisms capable of production ofsurfactant materials.

Surface active agents are used in soaps and industrial cleaners, intertiary oil recovery, in flotation, in emulsions, in foods, etc.Synthetic surfactants tend to dominate the market, however in recentyears microbes have been found to produce surfactants. Many of thesebiological surfactants have the advantage of being biodegradable,possessing low toxicity and high specificity for certain types of uses.

U.S. Pat. No. 3,997,398, J. E. Zajic and E. Knettig, describes in detailthe production, testing and utility of surfactant materials ofmicrobiological origin.

The present invention is based on the discovery of a certain class ofmicrobes which can be grown by aerobic fermentation processes undercontrolled conditions to give materials of outstanding surfactantproperties, in high yields. The microbes are certain cultures from theArthrobacter-Corynebacterium-Nocardia Genera which are characterized bytheir ability to metabolize the protective waxy hydrocarbon materialfound naturally on the plant cuticles of water plants such as salvinia,to expose the plant to disease-causing infections. The cultures arerepresented by a culture first isolated by us and referred to herein asCorynebacterium salvinicum strain SFC.

Thus, according to one aspect of the present invention, there isprovided a process for microbiological production of surfactantmaterials, which comprises cultivating by aerobic fermentation, in agrowth promoting medium and under growth promoting conditions, and on acarbon-containing substrate, a microbial strain of a culture from thegenera Arthrobacter, Corynebacterium and Nocardia, said microbial strainbeing characterized by ability to metabolize the waxy protective coatingof the plant cuticles of Salvinia water plant under normal growthconditions for the microbial strain; to produce a surfactant material ofmicrobiological origin in said fermentation medium.

A specific preferred microbial strain, representative of those useful inthe present invention, is Corynebacterium Salvinicum strain SFC. This isbelieved novel and not previously disclosed. A viable sample of thisculture has been deposited in the International Collection ofPhytopathogenic Bacteria, University of California, Davis, and has beengiven the reference number ICPB 4312 (Cooper SFC).

It is preferred to use hydrocarbons as the carbon-containing substratein the process of the invention, especially liquid aliphatic paraffinichydrocarbons, straight chain or branched chain. Most preferably, thehydrocarbons have from about 6 to about 18 carbon atoms per molecule.Mixtures of hydrocarbons such as kerosene can be used also. In addition,the microbes will grow on other carbon substrates such as carbohydratesand sodium acetate, but normally with less desirable results in theprocess of the invention.

On a small scale, the Corynebacterium salvinicum culture used here canbe grown by aqueous culture in shake flasks (500 ml), carboys (16liter), or fermentors (28, 48 liters) for the production of surfactant.The aqueous cultivation medium is important. It should contain theproper balance of minerals, especially inorganic nitrogen, phosphate andpotassium as well as other minerals and trace minerals required forgrowth of cells. The energy source for growth is preferably kerosene oraliphatic hydrocarbons representing the homologous series: undecane,dodecane, tridecane, tetradecane, pentadecane, hexadecane, heptadecaneand waxy hydrocarbons. The pH range for this process varies from 4 to8.8 depending on the inorganic nitrogen source and buffer system used.The system is an aerobic process, thus either air or oxygen must besupplied in adequate amounts required for growth and product formation.Mixing is accomplished by shaking in small laboratory vessels, howeverin larger vessels the mixing required should be in the low to highturbulent range. (Reynolds number of 1000 to 10,000). The optimaltemperature is around 30° C., but high yields can be obtained over thetemperature range of 18° to 37° C.

In the process for producing the surfactants according to the presentinvention, it is preferred to use a concentration of hydrocarbon in therange from 0.5 to 9.0% (W/V). Inorganic salts beneficial to thesurfactant production of the system of the present invention includessodium nitrate, ammonium sulphate, ammonium chloride, ammonium nitrate,ammonium carbonate and urea. As disclosed herein below, inorganicnitrogen compounds are beneficial to the surfactant production in thepresent process, the preferred range of concentrations of inorganicnitrogen being from 0.1 to 4.0% (W/V), with the most preferred rangebeing from about 0.6 to 4.0% (W/V). It is also desirable to includevitamins and essential nutrients in the fermentation medium of thepresent invention, by adding yeast extract and nutrient broth. Mostbeneficial ranges of concentrations for these materials are 0.1-3%(W/V), with optimum results at 0.1-2.0% concentration (W/V).

According to a further aspect of the process of the present invention,it has been found that isonicotinic acid hydrazide inhibits productionof surfactant. This material is available under the trade name"Isoniazid". It is used in treatment of tuberculosis, a disease causedby the microbe Mycobacterium tuberculosis. It specifically inhibitsmycolic acid production. However, it has been found that, if a smallamount of surfactant of the synthetic type, such as Tween 80 is added,the combination of both reagents, isonicotinic acid hydrazide and Tween80, increases the production of surfactant by the CorynebacteriumSalvinicum microbe several fold.

According to another aspect of the process of the present invention, theenzyme lysozyme can be used, for treatment of the cells produced in thefermentation process, either after fermentation is completed or duringthe fermentation, to obtain a further significant increase in surfactantconcentration. Preferred concentrations of lysozyme for this purpose arein the range 0.005-0.01%, (W/V).

According to a further aspect of the present invention, diethoxymethanecan be added to the fermentation broth, to obtain a beneficial effect onsurfactant production. Moreover, the total biomass produced in thepresence of diethoxymethane is increased. Its effect on cell morphologyis quite pronounced.

Culture Characteristics of Corynebacterium Salvinicum Strain SFC

The bacterial strain Corynebacterium salvinicum SFC was isolated from aplant source and was considered to be a plant pathogen. It is a Grampositive rod-form pleiomorphic organism that grows mainly in the oilphase. When in mid log phase its dimensions are about 2.5 μm ×1 μm. Itis non-motile and does not form chains. On 1.25% nutrient agar platesthe colonies are dry.

Corynebacterium salvinicum grows well on most aliphatic hydrocarbonsfrom C₁₀ -C₁₈. Hexadecane was required as a substate for good surfactantproduction. Taxonomic characteristics of this culture are summarizedbelow, and the growth characteristics on several substrates are shown inExample 2.

Morphological and Biochemical Characteristics of Corynebacteriumsalvinicum Strain SFC General Characteristics

Source: isolated from plant source as a pathogen

Gram stain: Gram positive

Size: about 2.5μ×1μ

Motility: non-motile

Colony on agar: on nutrient agar colonies are dry and star-shaped, onyeast glucose agar the colonies are dull, rough and creamy coloured.

Growth characteristics: optimum temperature 28°-30° C., no growth occursat 37° C. on plates. There is no anaerobic growth. No soluble pigmentwas produced.

Microscopic: 18 hr.-growth produces Gram positive diptheroid appearingrods, 72 hr.-growth has predominantly coccoid forms also with long formswhich appear to branch or divide by "snapping division".

Biochemical Characteristics

Nitrate reduction: positive

Citrate utilisation: positive

Glucose fermentation: negative (positive growth only)

Catalase: positive (very strongly) Starch hydrolysis: positive

    ______________________________________                                        Antibiotic Sensitivity Tests (Disk Test)                                                                Inhibition after                                    Antibiotic    Concentration                                                                             Four Days                                           ______________________________________                                        Penicillin    10 units    None                                                Penicillin G  10 units    7mm                                                 Tetracycline  30 mcg      6-8mm                                               Sulfisoxole   300 mcg     2.5cm                                               Methicillin   5 mcg       None                                                Kanamycin     30 mcg      7mm                                                 Ampicillin    10 mcg      8mm                                                 Cephaloridine 30 mcg      2.2-2.5cm                                           Clindamycin   2 mcg       None                                                Chloramphenical                                                                             30 mcg      4-6mm                                               Carbenicillin 100 mcg     8mm                                                 Erythromycin  15 mcg      1cm                                                 Gentamicin    10 mcg      2-4mm                                               Neomycin      30 mcg      2.5-3mm                                             Streptomycin  10 mcg      3-4mm                                               Polymyxin B   300 units   None                                                Erythromycin  15 mcg      8mm                                                 Isoniazid     50 mg/ml    Inhibited                                           ______________________________________                                    

The fermentation medium of choice contains all essential mineralsrequired for growth as well as hydrocarbon which is a source of energy.A portion of the hydrocarbon is also used in surfactant production. Italso induces surfactant production. One such media is described below:

    ______________________________________                                        Mineral Salts Medium                                                                            g/liter                                                     ______________________________________                                        NH.sub.4 SO.sub.4 2                                                           KH.sub.2 PO.sub.4 4                                                           Na.sub.2 HPO.sub.4                                                                              6                                                           MgSO.sub.4.7H.sub.2 O                                                                           0.2                                                         CaCl.sub.2.2H.sub.2 O                                                                           0.001                                                       FeSO.sub.4.7H.sub.2 O                                                                           0.001                                                       ______________________________________                                         Adjust to desired pH 6.5-8.0                                                  Hydrocarbon, Kerosene or aliphatic hydrocarbon 5 to 160 g                

The amounts of minerals required should be such that all requirementsfor growth and product formation are satisfied.

Biosurfactant Evaluation

Surfactants are known to decrease the surface tension when added towater. As the concentration of surfactant increases, the surface tension(ST) in dynes/cm (or mN/m) decreases to some minimal level until nofurther decrease is seen. Commonly this point ranges for mostsurfactants between 28-60 dynes/cm. The point or concentration at whichadditional increments of surfactant do not give additional decreases inST is known as the critical micelle concentration (CMC), and it is quitespecific for a given surfactant. If more than one surfactant is formed,several CMC values may be observed, particularly if each surfactant hasa different CMC.

The identification of the CMC values is important. In the process of theinvention, the actual yield of the surfactant or combination ofsurfactants can be obtained by a serial dilution technique, in which theproduction broth is diluted with water until the CMC value is reached.The greater the dilution required, the greater the concentration ofsurfactant produced and present in the broth. The apparent CMC of thewhole fermentation broth is a measure of the concentration of surfactantpresent in the broth. To effect measurements and obtain CMC values forthe whole broth, the whole fermentation broth may be dilutedconsecutively by a factor of 2 or 1.5, and surface tension measured andplotted as a function of the log₁₀ of the broth concentration. With anovel substance or one of which the composition is not known withprecision, the concentration of surfactant in absolute units cannot bedetermined. It is however possible to obtain figures of concentration ofsurfactant in units of the critical micelle concentration, which isrelated to the absolute concentration through an arbitrary constant. Thereciprocal of the dilution required to reach the CMC indicates theconcentration of surfactant in the whole broth in units of the CMC ofthat surfactant. Thus the reciprocal of dilution value at CMC is used asa measure of the concentration of surfactant times the CMC value of thesurfactant or surfactants.

Surface tensions of the whole fermentation broths were determined usinga Fisher Autotensiomat, which is a modified deNuoy surface tensionmeterwith a motorized sample stage and a strain gauge which measures tensionon the platinum ring. Output is directly in dynes/cm. The platinum ringis pulled upwardly through the aqueous solution, recording a plot ofdisplacement against tension. The maximum tension value on the curve,which is obtained as the ring passes through the liquid surface, is thesurface tension value.

The results are given in Table II.

The process and product of the present invention are further illustratedin the following specific examples.

EXAMPLE 1

The more important cultures developed and used for biosurfactantproduction in this study were taken and grown in a mineral salts mediumwith either kerosene or hexadecane and the concentration of surfactantproduced, in terms of the most concentrated surfactant, evaluated. Thedata, along with the Gibbs surface excess, is shown below.

    ______________________________________                                                         Surfactant                                                                             Gibbs                                                                Concentra-                                                                             Surface                                                              tion*    Excess                                                               × CMC                                                                            ρ moles/cm.sup.2                                ______________________________________                                        Corynebacterium salvinicum (SFC)                                                                 30,000       466                                           Corynebacterium lepus                                                                            3,030      2,100                                           Corynebacterium fasciens                                                                         75         1,050                                           Corynebacterium hydrocarboclastus                                                                8          1,300                                           Corynebacterium xerosis                                                                          7            800                                           Nocardia erythropolis                                                                            150        1,000                                           ______________________________________                                         *maximum value observed                                                  

EXAMPLE 2

Normally kerosene is the preferred source of hydrocarbon for surfactantproduction, however all of the aliphatic hydrocarbons can be used in theproduction of surfactant. The following hydrocarbons were tested at aconcentration of 3% (volume hydrocarbon to volume of aqueous medium),however they can be added over a wide range of concentrations and giveslightly better or poorer results. The pH used was 7.0.

    ______________________________________                                                      Surfactant    Surface or Inter-                                        Biomass                                                                              Concentration facial Tension*                                          g/l    × CMC.sub.1                                                                      × CMC.sub.2                                                                      ST     IT                                     ______________________________________                                        decane    .922    161      83     30     1.0                                  undecane  .874    27       16     34     1.0                                  dodecane  .804    74       16     31     2.0                                  tridecane                                                                              1.152    76       37     30     1.0                                  tetradecane                                                                            1.312    80       44     29     1.5                                  pentadecane                                                                            1.272    43       15     31     3.0                                  hexadecane                                                                             ND       434      98     ND     ND                                   ______________________________________                                         *Interfacial tension measured against                                         ND: Not determined                                                       

Other carbon sources also support growth of Corynebacterium salvinicumSFC. The growth and surfactant production obtained with 5 of these isgiven below. Sodium acetate is a poor carbon source for surfactantproduction, while hexadecane is an excellent source. Combination ofthese 2 substrates shows that sodium acetate is not an inhibitor ofgrowth or surfactant production; indeed, additional biomass can beobtained with the addition of sodium acetate to a hexadecane-basedculture medium.

Growth of Corynebacterium salvinicum Strain SFC on various carbonsources at 25°±2° C. in 500 ml Erlenmeyer shake flasks. Duration ofgrowth was 3 days.

    ______________________________________                                                                    Surfactant                                                        Total Biomass                                                                             Concentration                                     Carbon Source   (gm/l)      × CMC                                       ______________________________________                                        Fructose, 3% w/v                                                                              1.62        1.5                                               Glucose, 3% w/v 2.00        2.2                                               Sodium acetate, 3% w/v                                                                        0.65        2                                                 Sodium acetate, 1.5% w/v                                                      plus hexadecane, 1.5% v/v                                                                     4.38        82                                                Hexadecane, 3% v/v                                                                            2.80        95                                                ______________________________________                                    

EXAMPLE 3

Since hexadecane gave such high yields of surfactant, it wasinvestigated over a much broader range of concentrations. The standardmineral salts broth was used and the concentrations (%) of hexadecanetested were: 0.5, 1.0, 2.0, 3.0, 4.0, 5.0, 7.0 and 9.0% (v/v). Theinitial pH of the broth was 7.0. The concentration of surfactant interms of the CMC values is reported below.

    ______________________________________                                        Hexadecane      Surfactant Concentration                                      v/100 ml broth  × CMC.sub.1                                                                      × CMC.sub.2                                    ______________________________________                                        0.5             --        19                                                  1.0               434     98                                                  2.0             1,190    270                                                  3.0             6,670    800                                                  4.0             3,450    833                                                  5.0             *        833                                                  7.0             3,450    880                                                  9.0             5,000    869                                                  ______________________________________                                         *Sample spilled                                                          

The optimal concentration of hexadecane was 3.0% (v/v), however highlevels of surfactants were produced over the range of 1.0 to 9.0% (w/v)of hexadecane.

EXAMPLE 4

The selection of the source of nitrogen used for growth and productformation is an exacting requirement in a fermentation process. Theinorganic salts tested were ammonium sulfate, sodium nitrate, ammoniumchloride, ammonium nitrate, ammonium carbonate and urea. The range ofconcentrations tested were from 0.06 to 6.0% (w/v).

    __________________________________________________________________________                   Source of Nitrogen (percent)                                   Nitrogen       Concentration (g/v)                                            Compound Tested                                                                              .06                                                                             .1                                                                              .3 .6 1.0                                                                              2.0                                                                              3.0                                                                              4.0                                                                              5.0                                                                              6.0                                   (Initial pH 6.9)                                                                       Surfactant                                                                          Concentration of Surfactant in Terms of CMC ×            __________________________________________________________________________                   CMC                                                            NaNO.sub.3                                                                             CMC.sub.1                                                                             1.7                                                                             10 30 69 3.9                                                                              144                                                                              90 ND 58                                             CMC.sub.2                                                                             18                                                                              40 172                                                                              167                                                                              74 278                                                                              384                                                                              ND 175                                   (NH.sub.4).sub.2 SO.sub.4                                                              CMC.sub.1                                                                             --                                                                              122                                                                              952                                                                              666                                                                              1250                                                                             ND 526                                                                              ND 769                                            CMC.sub.2                                                                             --                                                                              33 71 113                                                                              454                                                                              ND 70 ND 118                                   NH.sub.4 Cl                                                                            CMC.sub.1                                                                             --                                                                              80 115                                                                              526                                                                              1520                                                                             1640                                                                             1920                                                                             ND 1850                                           CMC.sub.2                                                                             --                                                                              26 31 125                                                                              769                                                                              606                                                                              465                                                                              ND 333                                   NH.sub.4 NO.sub.3                                                                      CMC.sub.1                                                                             91                                                                              173                                                                              49 119                                                                              -- 2780                                                                             4000                                                                             ND ND                                             CMC.sub.2                                                                             37                                                                              34 17 28 -- 1000                                                                             1110                                                                             ND ND                                    NH.sub.4 CO.sub.3                                                                      CMC.sub.1                                                                             38                                                                              44 87 85 769                                                                              285                                                                              200                                                                              ND ND                                             CMC.sub.2                                                                             3.6                                                                             4.7                                                                              12 18 114                                                                              105                                                                              43 ND ND                                    Urea     CMC.sub.1                                                                           41                                                                              51                                                                              74 370                                                                              909                                                                              1490                                                                             250                                                                              ND ND ND                                             CMC.sub.2                                                                           8 8.7                                                                             9.9                                                                              103                                                                              169                                                                              307                                                                              39 ND ND ND                                    __________________________________________________________________________

All of the inorganic nitrogen sources reported herein supported somegrowth and production of both surfactants CMC₁ and CMC₂. The ammoniumchloride and ammonium nitrate systems were the most beneficial tosurfactant production at the initial pH used which was 6.9 in mostinstances. Urea and ammonium sulfate were next best and sodium nitrateand ammonium carbonate gave the poorest results. Since salts of sodiumnitrate tend to increase the pH during utilization of nitrate and saltsof ammonium tend to decrease pH during their utilization, pH was chosenas a variable for further study. Nitrate appears to influence andincrease CMC₂ much more than it does CMC₁.

EXAMPLE 5

Vitamin and essential nutrient supplement.

Yeast extract and nutrient broth contain a number of complex vitamins,some minerals and even some amino acids which are required for growth.Both of these were added over a specified range (0.3-6.0% wt/v) to themineral salts-kerosene medium. The initial pH was 7.0.

    __________________________________________________________________________    Complex Nutrient                                                                       Surfactant                                                                          Concentration Tested (%, wt/v)                                 Supplement                                                                             Synthesized                                                                         .06                                                                              .1 .3 .6 1.0                                                                              2.0                                                                              3.0                                                                              4.0                                                                              5.0                                                                              6.0                                 __________________________________________________________________________    Yeast Extract                                                                          CMC.sub.1                                                                           454                                                                              1190                                                                             1920                                                                             487                                                                              1960                                                                             323                                                                              606                                                                              ND ND ND                                           CMC.sub.2                                                                           139                                                                              274                                                                              159                                                                              117                                                                              147                                                                              164                                                                              294                                                                              ND ND ND                                  Nutrient Broth                                                                         CMC.sub.1                                                                           ND ND 1587                                                                             1492                                                                             1754                                                                             2440                                                                             200                                                                              380                                                                              217                                                                              208                                          CMC.sub.2                                                                           ND ND 645                                                                              357                                                                              556                                                                              488                                                                              64 153                                                                              71 84                                  __________________________________________________________________________     ND: Not determined                                                       

Nutrients of both these types proved to be beneficial for surfactantproduction. The range being most stimulating was 0.1 to 2.0% (wt/v).Nutrient broth increased the production of CMC quite effectively.

EXAMPLE 6

The initial pH of the broth before inoculation with culture SFC wasadjusted respectively to 4.8, 5.4, 5.8, 6.3, 6.6, 8 and 9. This culturesynthesizes two surfactants which can be identified by dilution to theirspecific CMC values. The broth medium used contained mineral salts andan optimum level of ammonium nitrate.

    ______________________________________                                        Biomass         Surfactant Concentration                                      pH     g/l          × CMC.sub.1                                                                        × CMC.sub.2                              ______________________________________                                        4.8    2.78         18,900     4,760                                          5.4    2.49         37,000     6,700                                          5.8    2.45         9,090      3,800                                          6.3    1.45         75         37                                             6.6    .177         6.8        5.0                                            7.0    ND           270        ND                                             8.0    ND           243        ND                                             9.0    ND           243        ND                                             ______________________________________                                         ND: Not determined                                                       

The optimal pH range appears to be between 4.8 and 5.8 but cannot berestricted within these levels. Maximum production occurred at pH 5.4.

EXAMPLE 7

Isonicotinic acid hydrazide (INH) (trade name: isoniazid) is used intreating tuberculosis, a disease caused by Mycobacterium tuberculosis.It specifically inhibits mycolic acid production. A test was set up inwhich INH was added to fermentation flasks at concentrations of 0, 0.1,0.3, 0.5, 1.0, 3.0, and 5.0×10⁻⁴ g/ml. A control series was run in whichno INH was added. A third series was completed in which a 0.01%concentration of Tween 80 (a synthetic surfactant) was added to eachvessel receiving INH. The results are summarized as follows:

    ______________________________________                                                  Dilution Required to give 65 dynes/cm                                                         Experimental + .01%                                 INH × 10.sup.-4 g/ml                                                                Experimental  (w/v) Tween 80                                      ______________________________________                                        0           5.06          9.49                                                .1          3.3           17.08                                               .3          2.0           11.39                                               .5          1.5           9.49                                                1.0         2.0           7.59                                                3.0         1.5           <1                                                  5.0         1.0           <1                                                  ______________________________________                                    

0.01% Tween 80 requires a dilution of <1 to give 69 dynes/cm.

INH was shown to inhibit surfactant prodution at all levels tested. Thefirst important discovery here is that a small amount of Tween 80, i.e.0.01%, can double surfactant production by Corynebacterium salvinicum.Tween 80 (0.01%) not only reverses the effect of INH at concentrationsup to 1×10⁻⁴ g/ml, it also stimulates the synthesis of surfactant by themicrobe at concentrations from 0.1 to 1.0×10⁻⁴ g/ml. The improvedsynthesis of surfactant was as large as 3 times that of the control (noINH, no Tween 80). This observation is an important method of increasingthe production of surfactant by cultures which produce mycolic acids,mycolic acid derivatives or related compounds.

EXAMPLE 8

The effect of the enzyme lysozyme on the growth and surfactantproduction by SFC was investigated. Lysozyme concentrations of 0.005%and 0.010% (w/v) were used, but the incubation was at 30° C. instead of25°±2° C. The surfactant concentration with 0.010 (w/v) lysozyme (addedto the flask) is about 1850×CMC compared to the 800×CMC obtained withthe control sample. However, it was also shown that with SFC growing inthe presence of 0.005% (w/v) lysozyme the surfactant concentrationincreases by a factor of more than 4 to a very high value of around3,200×CMC. The concentration value obtained with 0.01% lysozyme inminimal medium without culture was less than 1×CMC. This value is anaverage of two separate but duplicate studies. The duration of growth inthese studies was 3 days. The cell morphology changed quite dramaticallywith introduction of lysozyme. The cells rounded up and formedspheroplasts with both 0.005% and 0.01% lysozyme concentrations.However, the biomass values with 0.005% lysozyme were not much differentfrom control, but with 0.01% lysozyme decreased by about 20%, indicatingsome lysis of cells. A batch fermentation of the surfactanthyperproducing SFC strain was carried out in a 14 liter New Brunswickfermentor. Again, 3% hexadecane was used as the carbon and energy sourceand fermentation was carried out at 30° C. An increase in surfactantconcentration as well as increases in total biomass concentration anddecreases in hydrocarbon concentrations and O₂ consumption wereobserved. Surfactant concentration peaked after about 43 hours offermentation. The hexadecane concentration dropped rapidly after about32 hours of fermentation. The highest biomass value reached was about5.5 gm/l. A peak in surfactant concentration of about 1300×CMC wasattained while the hydrocarbon concentration was reduced by about 73%during the course of the fermentation (about 70 hours). The doublingtime of the microorganism was about 4 hours (specific growth rate μ=0.17hrs.⁻¹).

EXAMPLE 9

The alkane oxidation inducer diethoxymethane (DEM) increases theproduction of surfactant by SFC. The concentration of surfactantproduced was increased from 50×CMC to 65×CMC with the introduction ofDEM. The concentration of DEM required for this ranges from 0.05 μg/mlto 5 μg/ml. However, the effect on cell morphology was quite pronounced.The cells tended to be even in size but in large aggregations. The totalbiomass increased by about 15% with the introduction of DEM.

EXAMPLE 10 Emulsification properties of lipid extract fromCorynebacterium salvinicum, Strain SFC

Emulsions were produced using the lipid extract emulsifier from SFC incombination with either an aliphatic hydrocarbon (n-hexadecane), anaromatic hydrocarbon (benzene) or wax. The emulsion drops were observedunder the microscope. The emulsified hexadecane droplets ranged in sizefrom about 1.5 μm to about 15 μm. The drops were found to be quitestable over several hours. Benzene is also easily emulsified intodroplets. When viewed under the microscope, it was shown that the dropsformed tend to aggregate into clumps. Such clumps have also been notedbefore with other aromatics such as p-xylene. It is believed that thisaggregation makes aromatic hydrocarbon emulsions more stable. The waxemulsions show very unique features. The droplets are generally largerwith bits of solidified wax inside them. This gives the appearance ofmultiple phase emulsions. The wax emulsions are quite stable for severalhours.

When both aliphatic and aromatic emulsions were observed under themicroscope using polarised light, presence of liquid crystalline regionsin the emulsion drops were found. Symmetrical "illuminated" liquidcrystalline regions of a hexadecane droplet were observed. The presenceof these liquid crystalline regions in benzene droplets are easilyshown. Wax droplets did not show these regions.

The presence of liquid crystalline regions signifies excessconcentration of the emulsifying agent at the interface. It iscorrelated with increased stability of the emulsions.

The number of droplets with liquid crystalline regions was found to beslightly higher with the aromatic hydrocarbon than with the aliphatic(32% compared to 26% on averabe). It is believed that this is the firsttime that liquid crystalline regions in emulsion droplets have beenprepared using microbial emulsifiers.

The presence of liquid crystalline regions was also noted when aliquotsof hydrocarbon fermentation broth were observed microscopically underpolarised light. Preparation can be made to show the "illuminated"crystalline regions in a fermentation broth of strain SFC grown onhexadecane for 3 days. The presence of cells inside the hydrocarbonphase are seen. The degree of crystallinity is proportional to thedegree of "illumination" and regions of heavy crystallinity can beobserved. It was observed that the liquid crystalline regions were moreprevalent in the mid to late log phase broth than in the early log phasebroth, suggesting the presence of excess emulsifier in the mid to latelog phase. Dilution of the broth below the CMC value resulted in amarked reduction in the presence of liquid crystalline areas. Thisoccurrence of liquid crystalline regions in fermentation broths has notbeen reported before.

A study of the effect of carbon chain length upon emulsification usingthe lipid extract from SFC revealed a curious picture. There isgenerally an increase in emulsification with chain length except fordips with C₇ and C₁₀, and emulsification levels off after C₁₄. Theseobservations are quite different from the ones with the emulsifiers ofother cultures and from those reported elsewhere. The results wereobtained consistently and may reflect the chemical nature of theemulsifying agent(s) produced by SFC.

The PIT (Phase Inversion Temperature) value is a characteristic of asurfactant. An attempt was made to find the PIT value of the lipidextract from SFC. The inversion does not take place until a temperatureof 100° C. Thus the lipid extract had a PIT value between 99°-100° C.This is a very high value, particularly for a biological emulsifier, andpoints to the usefulness of this emulsifier for formulating emulsions athigh temperatures.

The importance of contact angles in studying surface phenomena isbecoming increasingly evident. Thus the relation of contact angles topathogenicity among microbes has been emphasized by previous work in thearea. Contact angles also have relevance in hydrocarbon fermentationswhere they determine the ability of a microbe to utilize hydrocarbons assubstrates for growth and maintenance. In this case, the role played byemulsifiers produced by the microbe is very significant. Contact angleswere measured between the various phases involved in hydrocarbonfermentation involving Corynebacterium salvinicum Strain SFC as well asCorynebacterium xerosis sp. Contact angle for both cultures, lipidextract, etc. are shown in the example below. As can be seen, thecontact angles involving the lipid extracts were very small or zero.Note also the reduction in contact angle between the cells andhexadecane when the lipid extract was dissolved in hexadecane. A smallercontact angle between the hydrocarbon containing the emulsifier andwater would permit better emulsification. The comparisons with use ofcommercial emulsifiers show the potency of the biological emulsifiers.Using this method one can, perhaps, determine the potential capabilityof a microbe to grow on certain hydrophobic substrates, and alsodetermine the influence of surfactants upon this capability and thus therelative ability of the microbial biosurfactant to wet biologicalsurfaces.

    ______________________________________                                        A. Contact angle measurements with/without lipid extract                      emulsifier from Corynebacterium salvinicum Strain SFC                         Surfaces              Contact Angle (°)                                ______________________________________                                        1.   Cells - hexadecane   23                                                  2.   Cells - hexadecane + lipid extract                                                                 0                                                   3.   Cells - lipid extract                                                                              0                                                   4.   Glass - lipid extract                                                                              1                                                   5.   Glass - hexadecane   30                                                  6.   Cells - hexadecane + Tween 80                                                                      0                                                   7.   Cells - supernatant  2                                                   8.   Glass - supernatant  10                                                  ______________________________________                                    

Index

The supernatant is the cell-free culture fluid from the fermentationbroth of the microbe growing on 3% hexadecane for 3 days.

    ______________________________________                                        B. Contact angle measurements with/without lipid extract                      emulsifier from Corynebacterium xerosis sp.                                   Surfaces              Contact Angle (°)                                ______________________________________                                        1.   Cells - hexadecane   24                                                  2.   Cells - hexadecane + lipid extract                                                                 1                                                   3.   Cells - lipid extract                                                                              0                                                   4.   Glass - lipid extract                                                                              2                                                   5.   Glass - hexadecane   30                                                  6.   Cells - hexadecane + Tween 80                                                                      0                                                   7.   Cells - hexadecane + Span 20                                                                       1                                                   8.   Cells - supernatant  2                                                   9.   Glass - supernatant  9                                                   ______________________________________                                    

Index

The supernatant is the cell-free culture fluid from the fermentationbroth of the microbe growing on 3% hexadecane for 3 days.

A study was made to examine the surface active components ofCorynebacterium salvinicum. The analysis was completed by making thinlayer chromatographic analyses for various components. These were:

    ______________________________________                                        Corynebacterium sp.                                                                         phosphatidylethanolamine, phos-                                 SFC lipid extract                                                                           phatidyl serine, phosphatidyl                                                 glycerol, 1,2- and 1,3-diglycerides,                                          triglyceride (high concentration),                                            fatty acid/ester, a fluorescent                                               component and two unknown                                                     components (digalactosyl diglyceride?).                         ______________________________________                                    

What we claim is:
 1. A process for microbiological production ofsurfactant materials, which comprises cultivating by aerobicfermentation, in a growth promoting medium and under growth promotingconditions, and on a carbon-containing substrate, the microbial strainCorynebacterium Salvinicum strain SFC, said microbial strain beingcharacterised by ability to metabolize the waxy protective coating ofthe plant cuticles of Salvinica water plant under normal growthconditions for the microbial strain, to produce a surfactant material ofmicrobiological origin in said fermentation medium, and recovering thesurfactant material so produced from said fermentation medium.
 2. Theprocess according to claim 1 wherein the fermentation broth includes amixture of isonicotinic acid hydrazide and Tween 80, in effectiveamounts to increase the surfactant production.
 3. The process accordingto claim 1 wherein the enzyme lysozyme is added to the cellular materialproduced during fermentation, to increase surfactant concentration. 4.The process according to claim 1 wherein the fermentation broth includesdiethoxymethane.
 5. The novel surfactant material of lipid-likestructure possessing emulsification properties, resulting from theprocess of claim
 1. 6. The process of claim 1 wherein thecarbon-containing substrate is an aliphatic liquid hydrocarbon havingfrom about 6 to about 18 carbon atoms per molecule.